Laboratorio de Helmintología, Instituto de Biología, Universidad Nacional Autónoma de México, México, D.F., Mexico;

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1 Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2015, 62: 018 doi: /fp Research Article A new species of Wallinia Pearse, 1920 (Digenea: Allocreadiidae) in Astyanax mexicanus (Characidae) from Mexico revealed by morphology and sequences of the 28S ribosomal RNA gene Gerardo Pérez-Ponce de León 1, Ulises Razo-Mendivil 2, 1, Miguel Rubio-Godoy 2 and Anindo Choudhury 3 1 Laboratorio de Helmintología, Instituto de Biología, Universidad Nacional Autónoma de México, México, D.F., Mexico; 2 Instituto de Ecología, Red de Biología Evolutiva, Xalapa, Veracruz, Mexico; 3 Division of Natural Sciences, St. Norbert College, De Pere, Wisconsin, USA Abstract: Wallinia mexicana sp. n. is described from the Mexican tetra, Astyanax mexicanus (De Filippi) (Characidae Weitzman), from two localities in northern Mexico. The new species can be distinguished from the two congeneric species, described from small-bodied characids in South and Central America, mainly by the posterior extent of the vitelline follicles (halfway between the posterior testis and the end of the caeca), by having a larger oesophagus, testes that are always oblique, and eye-spot remnants. The distinct status of sister species of W. chavarriae Choudhury, Hartvigsen et Brooks, 2002 described from characids in northwestern Costa Rica. Additionally, genetic divergence between these congeners reached 3.3%, a value higher than that observed for closely related species pairs of Magnivitellinum simplex Kloss, 1966 and Creptotrematina aguirrepequenoi Jiménez-Guzmán, 1973 in Astyanax mexicanus from Durango and San Luis Potosi states, respectively, are corrected. Keywords: Within Characiformes, a widespread tropical order of mately species (Mirande 2010). Within this family, the genus Astyanax Baird et Girard comprises the most diverse assemblage of more than 107 recognised species (Ornelas-García et al. 2008). In Mexico, nearly 41 species of characids currently distributed there (Pérez- Ponce de León and Choudhury 2010), and new host and distributional records, and even new species of helminths, continue to be reported on a regular basis (e.g. Mendoza-Franco et al. 2013, Pérez-Ponce de León et al. 2013, Aguilar-Aguilar et al. 2014, Razo-Mendivil et al. 2014a). Since characids are essentially Neotropical, phylogenetic analyses of members of their core helminth parasite fauna (sensu Pérez-Ponce de León and Choudhury 2005) are prime targets to help clarify patterns and processes that have shaped host associations and biogeography, and particularly for an analysis of host-switching and vicariance However, the diversity of the helminth parasite fauna in this host group needs to be well documented and the use of a DNA-taxonomy approach has shown that not all the species have been described yet. in river drainages of northern Mexico, two species of allocreadiids were reported from the Mexican tetra, Astyanax mexicanus (De Filippi), namely Magnivitellum simplex Kloss, 1966 and Creptotrematina aguirrepequenoi Jiménez-Guzmán, 1973 (see Pérez-Ponce de León et al. 2010, 2013). We recently sequenced the D1 D3 domains of the 28S rrna gene of specimens from that study and, on comparing sequences with those available for M. simplex, C. aguirrepequenoi and another closely similar allocreadiid, Wallinia chavarriae Choudhury, Hartvigsen et Brooks, 2002, realised that the aforementioned records of M. simplex and C. aquirrepequenoi may be in error. Reexamination of specimens from the original studies con- Address for correspondence: G. Pérez-Ponce de León, Instituto de Biología, UNAM, Ap. Postal , México, D.F., C.P , Mexico. Phone: ; Fax: ; ppdleon@ib.unam.mx ZooBank number for article: urn:lsid:zoobank.org:pub:1acd4866-f111-4fc7-87bb-b76a591006ee This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 species of Wallinia Pearse, Here we use morphological and molecular data to argue for the recognition of a new species of Wallinia. MATERIALS AND METHODS Species previously reported as Magnivitellinum simplex were collected from Astyanax mexicanus in the Río Covadonga, in Peñon Blanco, Durango State, in These specimens were deposited in the Colección Nacional de Helmintos, Instituto de Biología, the Colección Nacional de Helmintos, Mexico City, Mexico (CNHE), under accession numbers CNHE Prevalence and mean intensity of infection were 50% (2/4) and 5.5 digeneans per infected host, respectively. Two specimens - Creptotrematina aguirrepequenoi at the time were collected from A. mexicanus in the Río Huichihuayán, San Luis Potosí State, in 2009 and were deposited at the CNHE under accession number CNHE Prevalence and mean intensity of infection were 21% (4/19) and 1.8 digeneans per infected host, extraction. hot (steaming) 4% formalin, stained with Gomori s trichrome, a graded ethanol series, cleared in methyl salicylate and mounted as permanent slides using Canada balsam. Specimens are deposited in CNHE, Universidad Nacional Autónoma de México (UNAM), one specimen is deposited in the helminthological in micrometres, unless otherwise stated, with the range followed by the mean in parentheses. Drawings were made with the aid of a drawing tube attached to an Olympus BX51 microscope. For morphological comparisons, museum specimens were examined as follows. From the CNHE: Creptotrematina aguirrepequenoi ex Astyanax mexicanus, Cuatro Ciénegas, Coahuila (CNHE 8416); Magnivitellinum simplex ex Astyanax aeneus (Gunther), Río Ayuquila (CNHE 4787), ex A. mexicanus, Río Acamulco, Pánuco (CNHE 4840), ex A. fasciatus (Cuvier) (= A. aeneus), Cenote Dos Bocas, Quintana Roo (CNHE 2684), ex Ariopsis felis (Linnaeus), Ramonal in Rio Hondo, Quintana Roo (CNHE 2846), ex A. fasciatus (= A. aeneus), Torsuani, Nicaragua (CNHE 4195). From the Smithsonian National Museum of Natural History (NMNH), Washington, D.C. (formerly United States National Parasite Collection USNPC): Wallinia valenciae Pearse 1920, ex Geophyrocharax valenciae Eigenmann, Lake Valencia, Venezuela (USNPC 7569) (holotype); Wallinia chavarriae ex Bryconamericanus sceloparius Regan, Quebrada Limonal, Guanacaste, Costa Rica (USNPC 91364, holotype), ex A. aeneus, Río Sapoa, Guanacaste, Costa Rica (USNPC ), and Harold W. Manter Laboratory of Parasitology, Lincoln, Nebraska (HWML ). quencing, phylogenetic analyses and sequence divergence values follow those recently described by Razo-Mendivil et al. (2014a). Sequences generated in this study were deposited in the Gen- Bank database under accession numbers KJ KJ Sequences of the 28S rrna gene of the new species of Wallinia were compared with sequences recently analysed by Razo-Mendivil et al. (2014a) for testing the phylogenetic position of the sequenced specimens within the Allocreadiidae Looss, 1902, and for obtaining genetic divergence values through uncorrected p distances. RESULTS Wallinia mexicana sp. n. Figs. 1 5 ZooBank number for species: urn:lsid:zoobank.org:act:9a6982bc-b23f- 4B77-8ED1-360C8F8B7CCA Description (based on 12 whole-mounted specimens): Allocreadiidae. Body mm (2.88 mm) long, with maximum width (730) in post-testicular region where caeca end; anterior extremity bluntly rounded. Remnants of eye-spots present at level of pharynx. Oral sucker (254) long, (290) wide. Ventral sucker (272) long, (285) wide; opening ovoid or rounded. Ratio of oral sucker length to ventral sucker length 1 : (1 : 0.93); ratio of oral sucker width to ventral sucker width 1 : (1 : 1.02). Prepharynx lacking. Pharynx muscular, (115) long, (135) wide. Oesophagus narrow, relatively long, (193). Cluster of gland-cells situated laterally on either side of pharynx and oesophagus, clearly visible in specimens stained with Gomori s trichrome. Caecal bifurcation short distance anterior to ventral sucker, (496) from anterior end of body; caeca terminate blindly halfway between posterior testis and posterior end of body, at (659) from posterior end; left or right caecum occasionally longer. Testes two, always oblique, rounded or ellipsoidal, smooth; anterior testis (272) long, (198) wide; posterior testis (321) long, (195) wide, at mm (993 mm) from posterior extremity of body; intertesticular space (299). Cirrus-sac elongate, median, dorsal, overlapping ventral sucker and extending beyond its posterior margin, maximum width (69, n = 9); contains folded seminal vesicle continuous with narrower distal tubular portion, leading to indistinct, possibly ovoid pars prostatica obscured by surround- not observed. Genital pore median, immediately posterior to caecal bifurcation. Ovary smooth, not overlapping ventral sucker, (243) long, (158) wide, generally elongated or slightly triangular, with distinct distal region containing larger oocytes. Mehlis gland comprised of scattered glandcells obscuring ootype. Seminal receptacle ovoid, (144, n = 3) long, (133) wide, immediately posterior to ovary; Laurer s canal traversing dorsal side of body extending from level of caecal bifurcation/genital pore to halfway betweeen posterior margin of posterior testis and vitelline ducts unite immediately posterior to ootype to Folia Parasitologica 2015, 62: 018 Page 2 of 6

3 parasite was discovered. Figs Wallinia mexicana sp. n. from Astyanax mexicanus Fig. 1. Fig. 2. Ventral view of cirrus sac. Fig. 3. Dorsal view of cirrus-sac (from different specimen than in Fig. 2). Fig. 4. Female reproductive complex (with representative oocytes from different regions of the ovary). Fig 5. Operculate egg. form vitelline reservoir. Uterus tubular, passing posteriorly between intertesticular space or alongside testes, forming ular space and extending to posterior end of body. Eggs numerous, ovoid, operculate, embryonated, (56) long, (32) wide (n = 48). Excretory vesicle narrow, I-shaped, reaches level of anterior testes; excretory pore dorsoterminal. Type host: Astyanax mexicanus (De Filippi) (Actinopterygii: Characidae). Type locality: Río Covadonga at Peñon Blanco, Durango (24 43'56''N; '23''W). Other localities: Río Huichihuayán, San Luis Potosí (21 28'48''N; 98 58'2''W). Site of infection: Intestinal caeca, intestine. Specimens deposited: Holotype (complete wholemounted specimen, CNHE 8453); paratypes: 13 specimens from Río Covadonga (CNHE ); 6 specimens from Río Huichihuayán (CNHE 7411); 2 paratypes at the Institute lic (IPCAS D-710). mexicana refers to both the country and the host species (Astyanax mexicanus), where the Remarks. Wallinia mexicana sp. n. is placed in Wallinia because it exhibits the diagnostic characters of this genus (Choudhury et al. 2002), including an unspined tegument, well-developed cirrus-sac and medium genital pore. When describing W. chavarriae, Choudhury et al. (2002) tentatively placed the species within the Macroderoididae Mc- Mullen, They differentiated the new species from W. valenciae, the type species of the genus, a parasite of Gephyrocharax valenciae in Lake Valencia, Venezuela, by using the original description (Pearse 1920), which appears to have been based on a single worm, and by examining the holotype (USNPC 7569), which is now on its side. Al- W. chavarriae appeared to differ distinctly from W. valenciae in possessing a larger oral sucker (relative to the ventral sucker) and in having vitelline follicles that are rounded rather than tubular. Unfortunately, other details, such as those of the cirrus-sac and of the female reproductive complex, were either not certainty in W. valenciae (see Choudhury et al. 2002). The new species we describe herein most closely resembles W. chavarriae; however, it can be distinguished by having vitelline follicles extending beyond the posterior border of the posterior testis, to halfway between the posterior testis and the end of caeca, by having a larger oesophagus (193 μm vs 99 μm), distinctly oblique testes (not in tandem or slightly oblique), and by having remnants of eye-spots, or even diffuse eye-spots in some specimens (although they might have been overlooked in W. chavarriae). Wallinia chavarriae was described from two morphologically similar small-bodied characids, Astyanax aeneus and Bryconamericanus scleroparius, from Area Conservacion de Guanacaste, Costa Rica. Alignment of the 28S rrna gene consisted of 23 sequences (14 representing species of allocreadiids and eight of members of other xiphidiatan genera considered as outgroups). Alignment consisted of nucleotide positions, with 314 parsimony-informative characters. Maximum likelihood and Bayesian inference analyses recovered identical phylogenetic trees, whereas maximum parsimony recovered three equally parsimonious trees and the consensus tree showed a polytomy formed by the genera Auriculostoma Scholz, Aguirre-Macedo et Choudhury, 2004, Wallinia and Creptotrematina Yamaguti, 1953 (trees not shown). Maximum parsimony analysis recovered three equally parsimonious trees with a length of steps, a consistency index of 0.61, and a retention index of The representatives of the genus Auriculostoma were nested together with those of Wallinia and Creptotrematina (see Fig. 6) forming what Razo-Mendivil et al. (2014a) described as the characid clade for this group of allocreadiids that occur in the Neotropical biogeographical region. In that study the relationships among these allocreadiids were fully resolved, with Wallinia as the sister taxon of Creptotrematina and Auriculostoma. However, when the allocreadiid Paracreptotrema heterandriae Salgado-Mal- Folia Parasitologica 2015, 62: 018 Page 3 of 6

4 Fig. 6. Phylogenetic relationships (Bayesian inference) within the Allocreadiidae based on the 28S rrna gene showing the position of Wallinia mexicana sp. n. in bold. Numbers at the branches are posterior probability for Bayesian inference. in the phylogenetic analysis of the family, sister-group relationships change (see Razo-Mendivil et al. 2014b). In our phylogenetic analysis, the topology is similar to that obtained by the aforementioned authors when P. heterandriae is included and Margotrema bravoae Lamothe-Argumedo, 1970 is not included. This indicates that a more thorough sampling effort is needed to accomplish a robust phylogenetic analysis of lineages within the Allocreadiidae. gene was found between the two isolates of the new species found in Rio Huichihuayán and the one from Río Covadonga. Likewise, the genetic variation between W. chavarriae and the new species (3.3%) was even higher that that observed for other allocreadiid species pairs (Table 1). For instance, Razo-Mendivil et al. (2014a) obtained 2% variation of the 28S rrna gene between two sister species of allocreadiids, Auriculostoma totonacapensis Razo-Men- Godoy, 2014 and A. astyanace Scholz, Aguirre-Macedo et Choudhury, Actually, levels of genetic variation of this molecular marker have been used by several authors to differentiate species of digeneans (e.g. Pérez-Ponce de León et al. 2008, Tkach and Snyder 2008, Blasco-Costa et al. 2009, 2010). DISCUSSION The present study corrects previous reports of allocreadiids from Astayanax mexicanus and enables us to recognise previously undocumented diversity within the Wallinia spp. lineage. The new species we describe herein was originally recorded as two separate species. Pérez-Ponce de León et al. (2010) recorded the macroderoidiid M. simplex as a parasite of A. mexicanus in Río Covadonga in the Nazas River Basin, Durango State. Meanwhile, Pérez- Ponce de León et al. (2013) recorded C. aguirrepequenoi with specimens collected from A. mexicanus in Río Huichihuayán, San Luis Potosí, in the Panuco River Basin. Both records were wrong and sequence data obtained recently allowed us to recognise the error, resulting in the description of a new species of Wallinia here. Unfortunately, - (2002) argued that morphologically, the three species in these three genera are very similar. Additionally, all three species are parasites of the digestive tract of small-bodied characids such as Astyanax spp. and this has also misled previous efforts to identify these digeneans. Creptotrematina aguirrepequenoi and Magnivitellinum simplex has also been controversial. Folia Parasitologica 2015, 62: 018 Page 4 of 6

5 Table 1. Pairwise distance matrix of 28S rrna gene sequences of two isolates of Wallinia mexicana sp. n. (in bold) from two localities in northern Mexico with respect to other members of the Allocreadiidae. Species Allocreadium isoporum (Looss, 1984) - 2 Allocreadium lobatum Wallin, Auriculostoma astyanace Scholz, Aguirre-Macedo et Choudhury, Auriculostoma totocanapanensis Razo-Mendivil, Mendoza, Pérez et Rubio-Godoy, Bunodera sp Bunodera lucioperca (Müller, 1776) Crepidostomum cornutum (Osborn, 1903) Crepidostomum illinoense Faust, Creptotrema funduli Mueller, Creptotrematina aguirrepequenoi Jiménez-Guzmán, Megalogonia ictaluri Surber, Paracreptotrema heterandriae Salgado-Maldonado, Wallinia chavarriae Choudhury, Hartvigsen et Brooks, Wallinia mexicana sp. n. (Huichihuayán) Wallinia mexicana sp. n. (Covadonga) Yamaguti (1971) placed Magnivitellum simplex and Wallinia valenciae Pearse, 1920 in the Macroderoididae. Later, C. aguirrepequenoi was placed in the Bunoderidae Nicoll, 1914 by Jiménez-Guzmán (1973). This author also found M. simplex val, Both M. simplex and C. aguirrepequenoi were recorded from Astyanax fasciatus mexicanus (= A. mexicanus De Filippi) from the Presa Rodrigo Gómez in Nuevo León, within the Río Bravo (= Grande) basin in northeastern Mexico. Jiménez-Guzmán (1973) discussed the extent of morphological variation in the two species in terms of posterior extension of the vitelline follicles, testes position (oblique, tandem or symmetrical), absence of tegumental spines (in M. simplex) and absence of eye-spots in C. aguirrepequenoi. Thatcher (1993) placed Creptotrematina spp. and M. simplex in the Allocreadiidae Looss, Choudhury et al. (2002) argued that as unspined plagiorchiforms with a well-developed cirrus sac, a median genital pore and a tandem arrangement of ovary and testes in the hindbody, Wallinia spp. belonged in Allocreadiidae. However, the oblique placement of the testes, the laterally restricted vitellaria and the manner in which the uterus passes between the testes on its way to an extensive occupation of the post-testicular area in Wallinia spp., M. simplex, and Creptotrematina spp. are all suggestive of macroderoidid ses have unequivocally demonstrated that, at least Wallinia sp. and C. aguirrepequenoi, belong to the Allocreadiidae (Pérez-Ponce de León et al. 2007, Curran et al. 2011, Razo- Mendivil et al. 2014a). Wallinia mexicana sp. n. is the third species described in the genus and it was found in the characid A. mexicanus. The Mexican tetra has the most northern distributional range among characids, occurring in northern Mexico and southern U.S.A., in river basins such as the Bravo, Conchos, Nazas, Aguanaval, Mezquital, Tuxpan and Panuco (Miller et al. 2005). Species of Wallinia, like members of the allocreadiid genera Auriculostoma and Creptotrematina are part of the core parasite fauna of characids (see Pérez-Ponce de León and Choudhury 2005) and, as in the case recently described for Auriculostoma by Razo-Mendivil et al. (2014a), it seems that the genus has established a close phylogenetic association with characids in the Neotropical region. These two species appear to be the result of common speciation events that occurred after the isolation of A. mexicanus in the river basins of northern Mexico. Further studies where DNA sequences are obtained for isolates in different localities will determine if this is the only species of Wallinia in the Mexican tetra and in other characids in Mexico. Acknowledgments. We thank the following people for their help Rogelio Aguilar, David Hernández and Rogelio Rosas. Thanks are due to Omar Domínguez and Rodolfo Pérez for their help in CNHE, Mexico City, for the loan of specimens, and Laura Márquez, for the help with the use of the automated sequencer. This to GPPdL, CONACYT No to GPPdL and CB to MRG. AC wishes to thank St. Norbert College Faculty Development funds for partial support. REFERENCES : Helminth para- Chihuahuan desert of Mexico: inventory and biogeographical implications. Int. Zool. 9: P.D. 2009: Interrelationships of the Haploporinae (Digenea: Haploporidae): a molecular test of the taxonomic framework based on morphology. Parasitol. Int. 58: : Molecules and morphology reveal cryptic Folia Parasitologica 2015, 62: 018 Page 5 of 6

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